Enzyme containing granule

ABSTRACT

This invention relates to an enzyme containing granular composition comprising:  
     a) an enzyme containing core and  
     b) a protective substantially continuous layer or coating encapsulating the core comprising at least 60% of a water soluble compound, having a molecular weight below 500 grams per mole, a pH below 11 and a constant humidity at 20° C. of more than 81%.  
     The invention provides an improved stability of enzymes upon storage.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S.application No. 09/872,922 filed on Jun. 1, 2001, which is acontinuation of U.S. application Ser. No. 09/344,877, filed Jun. 25,1999, and claims, under 35 U.S.C. 119, priority of Danish applicationNo. PA 1998 00876, filed Jun. 30, 1999, and benefit of U.S. provisionalapplication No. 60/092,003, filed Jul. 8, 1998. Priority from U.S.application Ser. No. 09/344,877, filed on Jun. 25, 1999, is claimedunder 35 U.S.C. 120, the contents of which are fully incorporated hereinby reference

FIELD OF THE INVENTION

[0002] The present invention relates to an enzyme containing granuleprotecting the enzyme from inactivation when the granules are stored,i.e. improving the storage stability of the enzyme. The inventionfurther relates to a process for producing such a granule and to the useof the granule in a number of industrial applications such asincorporation of the granule in a detergent composition.

BACKGROUND OF THE INVENTION

[0003] The industrial use of enzymes, notably enzymes of microbialorigin, has become increasingly common. Enzymes are used in numerousindustries, including, for example, the starch-processing industry andthe detergent industry.

[0004] It is well known that enzymes upon storage is liable to bedegraded or inactivated by components (such as oxygen or bleachcomponents) from the surrounding matrix (such as a detergent), which arecapable of oxidizing or otherwise inactivating the enzyme. Further it iswell known that a low formation of enzyme-containing dust of granules isdesired.

[0005] Since the introduction of enzymes into the detergent industry, alot of effort has been devoted to improving the formulation of enzymeproducts by applying granulation and coating of the enzyme so as to bothprotect the enzyme from degradation as well as control enzyme dustformation.

[0006] Many granulates are composed of a core particle upon which anenzyme containing layer is added. The core may also in itself containenzyme. To apply the desired properties of this construction, e.g.color, dust formation, solubility rate, size, enzyme stability, physicalstrength etc. the core-enzyme construction is usually added additionallayers of coatings providing such properties.

[0007] Some coating layers described in the art are complexmulti-component compositions such as in:

[0008] WO 90/09440 which discloses an enzyme containing granulate 1) onecoating (or shell) comprising a binder (e.g. kaolin), a filler (e.g.inorganic salts), granulating agents (e.g. cellulose fibers providingphysical strength), and an enzyme and 2) a second dust suppressingcoating (mono-, di- or triglyceride).

[0009] DE 4322229 which discloses an enzyme containing granule with acoating comprising an inorganic pigment, an alcohol, an emulsifier, apigment dispersant and water.

[0010] JP 61162185 which discloses a process for production of an enzymecontaining granule comprising coating a core with a solution containingenzyme(s), sodium sulfate and optionally binders and coating agents.

[0011] Other coating layers apply polymers or even macroscopic particlesto gain improved properties of the granulate such as: WO 97/23606 whichdiscloses an enzyme containing granule comprising an outer coating ofpolyvinyl pyrrolidone, PVA or PEG.

[0012] WO 96/38527 which discloses an enzyme containing granulatedsubstance with a coating comprising water insoluble particles and abinder.

[0013] U.S. Pat. No. 5,324,649 which discloses an enzyme containinggranule comprising an outer coating of polyvinyl alcohol or a copolymer.

[0014] WO 93/07263 which discloses an enzyme containing granulecomprising an outer coating of vinyl (co)polymer.

[0015] WO 92/12645 which discloses an enzyme containing T-granulatecoated with high melting fat or wax.

[0016] WO 89/08694 which discloses an enzyme containing granulate with acoating comprising a mono- or diglyceride of a fatty acid.

[0017] DD 263790 which discloses a protease containing granule with acoating of skim milk and/or maltodextrin.

[0018] WO 87/07292 which discloses an enzyme containing granulate with acoating containing a copolymers of acrylic acid, and/or an filler and/ora plasticiser.

[0019] EP 193829 and U.S. Pat. No. 4,689,297 which discloses a processfor production of an enzyme containing particle comprising coating theparticle with a macromolecular, film forming water soluble or waterdispersible coating agent.

[0020] JP 58179492 which discloses an enzyme supporting particle with acoating of modified cellulose.

[0021] WO 89/08695 which discloses an enzyme containing particles with acoating containing clay.

[0022] Still other prior art disclosures apply non aqueous liquids ascoatings to gain improved properties of the granulate such as:

[0023] WO 96/16151 which discloses a enzyme containing granule with acoating of a non-aqueous liquid.

[0024] Some prior art disclosures mention use of agents in a coatinglayers providing a special functions upon dissolution of the granulatessuch as:

[0025] DE 4344215 which discloses an enzyme containing granule with acoating containing an inorganic Ag-corrosion inhibitor.

[0026] EP 206417 which discloses an enzyme containing granule with acoating containing an alkaline buffer salt having a pH of 7-11. Thebuffer salt may constitute 50-100% of the coating.

[0027] WO 93/07263 discloses an enzyme containing granule which containsa scavenger layer, preferably ammonium sulfate.

[0028] EP-415652-A2 as well as U.S. Pat. No. 5,093,021 describes enzymegranules coated with unsoluble and highly alkaline alkali metalsilicates optionally in combination with alkali metal carbonates.

[0029] Use of low molecular water soluble compounds in coatings inmoderate amounts have been disclosed within the art of formulatingstabile enzyme compositions usually as filler material. However, it hasnot been acknowledged that the amount and hygroscopicity of suchcompounds has essential impact on the stability of an enzyme in anenzyme granule coated with such compounds.

SUMMARY OF THE INVENTION

[0030] We have in our search for enzyme formulation with improved enzymestability surprisingly found, that an enzyme containing granule coatedor encapsulated by a continuous layer of a simple, predominantly watersoluble and low cost material with a high constant humiditysignificantly increases the storage stability of the enzyme, especiallyat high humidity conditions.

[0031] The present invention provides thus in a first aspect an enzymecontaining granule comprising:

[0032] a) an enzyme containing core and

[0033] b) a protective substantially continuous layer or coatingencapsulating the core comprising at least 60% of a water solublecompound, having a molecular weight below 500 grams per mole, a pH below11 and a constant humidity at 20° C. of more than 81%.

[0034] In accordance with the first aspect a second aspect of theinvention is a method for producing said enzyme containing granulecomprising coating said enzyme containing core with said coatingmaterial.

[0035] In accordance with the previous aspects further aspects of theinvention relates to applications of the enzyme containing granules,e.g. incorporation of the granule in a detergent or an animal feedcomposition or a baking composition and a cleaning method comprisingcontacting an object with an aqueous solution of the enzyme containinggranule.

DESCRIPTION OF DRAWING

[0036]FIG. 1 shows a cross section of an example of a granule accordingto the invention depicting different granule layers.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Definitions

[0038] The term “% RH” is used throughout the text, and in the contextof the invention the term is to be understood as the relative humidityof air. 100% RH is air saturated with water moisture at a fixedtemperature and % RH thus reflects the percent moisture saturation ofthe air.

[0039] The term “constant humidity” (in the context of the inventionsometimes abbreviated as CH) of a compound or substance is to beunderstood as the % RH of atmospheric air in equilibrium with asaturated aqueous solution of said compound in contact with the solidphase of said compound, all confined within a closed space at a giventemperature. This definition is in accordance with “Handbook ofchemistry and physics” CRC Press, Inc., Cleveland, USA, 58th edition, pE46, 1977-1978. Accordingly CH_(20° C.)=50% for a compound means thatair with a 50% humidity will be in equilibrium with a saturated aqueoussolution of the compound at 20° C. Accordingly the term constanthumidity is a measure of the hygroscopic properties of a compound.

[0040] The term “pH” of a compound in the context of the invention is tobe understood as the pH of a 10% w/w aqueous solution of the saidcompound.

[0041] The term “water soluble compound” in the context of the inventionis to be understood as a compound for which at least 0.1 grams of thecompound may dissolved in 100 g of water at 20° C., preferably at least0.5 g per 100 g water, e.g. at least 1 g per 100 g water.

[0042] The Protective Coating

[0043] Without being bound to this theory we believe that a coating witha high constant humidity value has at least one important functionalproperty: The coating inhibits moisture from entering the enzymecontaining core material. The coating is thus a barrier between thepotentially harmful matrix which surrounds the enzyme containinggranules (e.g. a detergent and/or air). For moisture and/or harmfulcomponents carried by the moisture to reach the enzyme within a granule,the moisture must pass through the coating (i.e. the moisture must beabsorbed on the outside and liberated on the inside surface of thecoating) before coming in contact with and inactivating the enzyme.Coatings with a high constant humidity (CH) thus provides a betterprotection of the enzyme than coatings with a lower constant humidity,i.e. the high humidity constant coating will inhibit moisture fromentering the granule at a broader ranger of humidity conditions (% RH)of the surrounding matrix. We believe that if as an example the % RH ishigher than the CH of the coating material the coating will absorbmoisture from the surrounding matrix and allow the moisture to betransported inside the granule.

[0044] For coatings allowing absorption of moisture from the surroundingmatrix, the rate of moisture absorption, and thus the damaging affectson the enzyme is believed to be further increased if the core materialalso readily absorbs the incoming moisture, which is the case for manyknown core materials. The moisture absorption process may thus beaccelerated by swelling or expansion of the core materials formingcracks, holes or disruptions in the coating, thus making moisture accessto the enzyme in the core even easier. Choosing a coating with aconstant humidity value higher than the expected % RH of the surroundingair thus effectively reduces this process.

[0045] Further a coating with a high constant humidity value is believedto inhibit entry of microorganisms thus reducing the possibility ofmicrobial growth within the granule.

[0046] As mentioned vide supra a suitable coating according to theinvention comprises at least 60% w/w of a water soluble compound, havinga molecular weight below 500 grams per mole, a pH below 11 and aconstant humidity of more than 81% w/w. Water soluble compounds having amolecular weight below 500 g/mole are usually much cheaper and easier tohandle with respect to coating processes than high molecular substancessuch as polymers. Preferred water soluble compounds should have a molarweight in the range of 30-500 g/mole preferably 75-400, e.g. 100-300g/mole.

[0047] Further a water soluble compound benefits release and/ordissolution of the enzyme upon introduction of the granule in an aqueousmedium (e.g. a cleaning or washing liquor) much more than insolublesalts such as calcium carbonate and minerals or inorganic compounds suchas kaolin and/or titandioxide. Accordingly a preferred water solublecompound is an inorganic salt, e.g. salts of sulfate, sulfite,phosphate, phosphonate, nitrate, chloride or carbonate or salts ofsimple organic acids (less than 10 carbon atoms e.g. 6 or less carbonatoms) such as citrate, malonate or acetate. Preferred cations in thesesalt are alkali or earth alkali metal ions, although the ammonium ion ormetal ions of the first transition series, e.g. Zinc may also be used.Especially alkali- or earth alkali metal salts of sulfate, sulfite,phosphate, phosphonate, nitrate, chloride or carbonate or salts ofsimple organic acids such as citrate, malonate or acetate are preferred.

[0048] A suitable solubility should be that at least 0.1 grams of thesalt in 100 g of water at 20° C., preferably at least 0.5 g per 100 gwater, e.g. at least 1 g per 100 g water. In a most preferred embodimentof the invention the solubility of the water soluble compound is atleast 10 grams or at least 20 grams of compound per 100 gram water at20° C. A high solubility is very advantageous as it benefits the coatingprocess as it lowers the amount of water needed to be evaporated afterdelivering the coating on the core. Further it is important that thecompound is dissolved in an aqueous phase before coating, because if acore is coated by applying the water soluble compound in dry form as aparticulate powder or as a slurry on the core, these particles will formchannels or openings in the coating allowing access of moisture to thecore.

[0049] The water soluble compound should also have a moderate pH inaqueous solution as extreme pH values of coating solution may corrodeequipment as well as being potentially dangerous to work with. Thus thewater soluble compound may be a slightly alkaline or slightly acidiccompound. Accordingly the pH of the water soluble compound should bebelow 11, preferably below 10, e.g. below 9, below 8 or even below 7,when measured as a 10% w/w aqueous solution of the water solublecompound. Some soluble salts like sodium carbonate has a very high pH(above 11) and may not be suitable as a coating material in the contextof this invention. Also bicarbonate salts may be unsuitable as theyalthough they have a lower pH tend to form carbon dioxide gas insolution which may interfere with the coating process.

[0050] Specific examples of suitable water soluble compounds of theinvention are Na₂HPO₄ (CH_(20° C.)=95%), Na₃PO₄ (CH_(25° C.)=92%) (NH₄)H₂PO₄ (CH^(20°0 C.=)93.1%), KH₂PO₄ (CH_(20° C.)=92%) ,Na₂SO₄(CH_(20° C.)=93%), K₂SO₄(CH_(25° C.)=99%), KHSO₄(CH_(20° C.)=86%), ZnSO₄ (CH_(20° C.)=90%) and citrate(CH_(25° C.)=86%). Sodium sulfate and sodium citrate are the mostpreferred water soluble compounds as they are cheap chemicals.

[0051] The coating comprises as said at least 60% w/w, e.g. 65% w/w or70% w/w of the water soluble compound, which preferably may be at least75% w/w, e.g. at least 80% w/w, at least 85% w/w, e.g. at least 90% w/wor at least 95% w/w. The coating may even essentially consist of thewater soluble compound. With due respect for maintaining a desiredsuitable constant humidity value for the total coating material minoramounts of other feasible compounds may be present in the coating suchas conventional coating materials. Examples of are, inter alia,described in the paragraph “additional coatings” given below. Otherexamples of conventional coating materials may be found in referencessuch as U.S. Pat. No. 4,106,991, EP 170360, EP 304332, EP 304331, EP458849, EP 458845, WO 97/39116, WO 92/12645A, WO 89/08695, WO 89/08694,WO 87/07292, WO 91/06638, WO 92/13030, WO 93/07260, WO 93/07263, WO96/38527, WO 96/16151, WO 97/23606, U.S. Pat. Nos. 5,324,649, 4,689,297,EP 206417, EP 193829, DE 4344215, DE 4322229 A, DD 263790, JP 61162185 Aand/or JP 58179492.

[0052] In a particular embodiment the coating may comprise minor amountsof a protective agent capable of reacting with a component capable ofinactivating (being hostile to) the enzyme entering the granule from thesurrounding matrix, i.e. before the component come into contact andinactivate the enzyme. The protective agent may thus e.g. be capable ofneutralizing, reducing or otherwise reacting with the hostile componentrendering it harmless to the enzyme. Typical components capable ofinactivating the enzyme are oxidants such as perborates, percarbonates,organic peracids and the like.

[0053] Protective agents may fall into several categories: alkaline orneutral materials, reducing agents, antioxidants and/or salts of firsttransition series metal ions. Each of these may be used in conjunctionwith other protective agents of the same or different categories.Examples of alkaline protective agents are alkali metal silicates,-carbonates or bicarbonates which provide a chemical scavenging effectby actively neutralizing e.g. oxidants. Examples of reducing protectiveagents are salts of sulfite, thiosulfite or thiosulfate, while examplesof antioxidants are methionine, butylated hydroxytoluene (BHT) orbutylated hydroxyanisol (BHA). Most preferred agents are salts ofthiosulfates, e.g. sodium thiosulfate. The amounts of protective agentin the coating may be 1-40% w/w of the coating, preferably 5-30%, e.g.10-20%.

[0054] In accordance with the concept of the invention the coating doesnot contain any enzyme, as the purpose of the coating is to protectenzymes encapsulated by the coating.

[0055] The protective effect of the coating depends on the thickness ofthe coating and the amount of coating relative to the rest of the enzymecontaining granule. An increased coating thickness provides a betterprotection of the enzyme, but at the same time result in increasedmanufacturing costs as well as a risk of poorer granule properties withrespect to enzyme solubility rate upon introduction of the enzymecontaining granule in an aqueous medium. For effective protection thecoating thickness also must be adjusted to the size of the core, e.g. toobtain a desired size of the finishes granule. Depending on the size ofthe core material the coating may be applied in 1-75% w/w of the weightof the coated granule to obtain a desired size of the coated granule.For small sizes of core material the coating may be applied in 50-75%w/w or 15-50% of the coated granule. Usually coatings constituting 2-20%w/w, preferably 3-10% w/w, e.g. 6% of the coated granule is howeverpreferred.

[0056] The coating should encapsulate the enzyme containing core byforming a substantially continuous layer. The layer or coating ispreferably homogenous in thickness and by substantially continuous ismeant that the core surface should have few or none uncoated areas.

[0057] The Core

[0058] The core contains the enzyme(s). Besides of the enzyme(s) thecore may be constructed in any way or of any material which provides thedesired functional properties of the core material, e.g. the core mayconsist of materials which allows readily release of the enzyme(s) uponintroduction to an aqueous medium. In one preferred embodiment the coreis constructed of a particulate carrier (I) with the enzyme absorbedand/or an enzyme containing layer (II) applied on the carrier surface,optionally comprising an enzyme protecting reducing agent. There mayeven be additional coating within the core material providing desiredfunctional properties of the core material. Another preferred core isthe so called T-granulate wherein enzyme and granulation material ismixed to form granules incorporating the enzyme distributed throughoutthe core such as described in U.S. Pat. No. 4,106,991 e.g. Example 1.Any conventional methods and non-enzyme materials may be used to preparethe core. Examples of known conventional cores and materials is, interalia, described in, U.S. Pat. No. 4,106,991 (in particular), EP 170360,EP 304332, EP 304331, EP 458849, EP 458845, WO 97/39116, WO 92/12645, WO89/08695, WO 89/08694, WO 87/07292, WO 91/06638, WO 92/13030, WO93/07260, WO 93/07263, WO 96/38527, WO 96/16151, WO 97/23606, U.S. Pat.Nos. 5,324,649, 4,689,297, EP 206417, EP 193829, DE 4344215, DE 4322229A, DD 263790, JP 61162185 A, JP 58179492.

[0059] As a particularly preferred embodiment of the invention the coremay be prepared by applying an enzyme layer onto a “placebo” carrier(enzyme-free carrier) according to the methodology described in U.S.Pat. No. 4,106,991. Optionally additional enzyme may be absorbed intothe surface of the carrier.

[0060] In a particular embodiment of the invention the enzyme containingcore may also comprise a protective agent as described for the coating,vide supra, preferably mixed with the enzyme in suitable amounts such as0.1-1% w/w of the coated granule, preferably 0.1-0.5% w/w, e.g. 0.33%w/w.

[0061] As described, supra, the core may through the coating absorbmoisture from the surrounding environment, a process which may cause thecore to swell resulting in crack formation in the coating and furthermoisture absorbance. The core may even in at high relative humiditydissolve and become fluid. Accordingly in order to provide furtherstabilization of the enzyme the core should preferably be a nonabsorbing core, i.e. the should only be able of absorbing less moisturethan 20% w/w of it own dry weight, preferably less than 10% w/w, e.g.less than 8% w/w or less than 5% w/w, measured at 75% RH at 20° C.

[0062] Enzymes

[0063] The enzyme in the context of the present invention may be anyenzyme or combination of different enzymes, which benefits from beinggranulated and thus be protected against a hostile environment in orderto be applicable for a specific use. Accordingly, when reference is madeto “an enzyme” this will in general be understood include combinationsof one or more enzymes.

[0064] It is to be understood that enzyme variants (produced, forexample, by recombinant techniques) are included within the meaning ofthe term “enzyme”. Examples of such enzyme variants are disclosed, e.g.,in EP 251,446 (Genencor), WO 91/00345 (Novo Nordisk A/S), EP 525,610(Solvay) and WO 94/02618 (Gist-Brocades Nev.).

[0065] The enzyme classification employed in the present specificationwith claims is in accordance with Recommendations (1992) of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology, Academic Press, Inc., 1992.

[0066] Accordingly the types of enzymes which may appropriately beincorporated in granules of the invention include oxidoreductases (EC1.-.-.-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC4.-.-.-), isomerases (EC 5.-.-.-) and ligases (EC 6.-.-.-).

[0067] Preferred oxidoreductases in the context of the invention areperoxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC1.1.3.4)], while preferred transferases are transferases in any of thefollowing sub-classes:

[0068] a) Transferases transferring one-carbon groups (EC 2.1);

[0069] b) transferases transferring aldehyde or ketone residues (EC2.2); acyltransferases (EC 2.3);

[0070] c) glycosyltransferases (EC 2.4);

[0071] d) transferases transferring alkyl or aryl groups, other thatmethyl groups (EC 2.5); and

[0072] e) transferases transferring nitrogenous groups (EC 2.6).

[0073] A most preferred type of transferase in the context of theinvention is a transglutaminase (protein-glutamine αglutamyltransferase; EC 2.3.2.13).

[0074] Further examples of suitable transglutaminases are described inWO 96/06931 (Novo Nordisk A/S).

[0075] Preferred hydrolases in the context of the invention are:Carboxylic ester hydrolases (EC 3.1.1.-) such as lipases (EC 3.1.1.3);phytases (EC 3.1.3.-), e.g. 3-phytases (EC 3.1.3.8) and 6-phytases (EC3.1.3.26); glycosidases (EC 3.2, which fall within a group denotedherein as “carbohydrases”), such as Δ-amylases (EC 3.2.1.1); peptidases(EC 3.4, also known as proteases); and other carbonyl hydrolases].

[0076] In the present context, the term “carbohydrase” is used to denotenot only enzymes capable of breaking down carbohydrate chains (e.g.starches) of especially five- and six-membered ring structures (i.e.glycosidases, EC 3.2), but also enzymes capable of isomerizingcarbohydrates, e.g. six-membered ring structures such as D-glucose tofive-membered ring structures such as D-fructose.

[0077] Carbohydrases of relevance include the following (EC numbers inparentheses): Δ-amylases (3.2.1.1) , Ε-amylases (3.2.1.2) , glucan1,4-Δ-glucosidases (3.2.1.3), cellulases (3.2.1.4),endo-1,3(4)-Ε-glucanases (3.2.1.6), endo-1,4-Ε-xylanases (3.2.1.8),dextranases (3.2.1.11), chitinases (3.2.1.14), polygalacturonases(3.2.1.15), lysozymes (3.2.1.17), Ε-glucosidases (3.2.1.21),Δ-galactosidases (3.2.1.22), Ε-galactosidases (3.2.1.23),amylo-1,6-glucosidases (3.2.1.33), xylan 1,4-Ε-xylosidases (3.2.1.37),glucan endo-1,3-Ε-D-glucosidases (3.2.1.39), Δ-dextrinendo-1,6-Δ-glucosidases (3.2.1.41), sucrose Δ-glucosidases (3.2.1.48),glucan endo-1,3-Δ-glucosidases (3.2.1.59), glucan 1,4-Ε-glucosidases(3.2.1.74), glucan endo-1,6-Ε-glucosidases (3.2.1.75), arabinanendo-1,5-Δ-L-arabinosidases (3.2.1.99), lactases (3.2.1.108),chitosanases (3.2.1.132) and xylose isomerases (5.3.1.5).

[0078] Examples of commercially available oxidoreductases (EC 1. - .- .-) include Gluzyme□ (enzyme available from Novo Nordisk A/S). Furtheroxidoreductases are available from other suppliers.

[0079] Examples of commercially available proteases (peptidases) includeEsperase□, Alcalase□, Neutrase□, Durazym□, Savinase□, Kannase, Pyrase□,Pancreatic Trypsin NOVO (PTN), Bio-Feed□ Pro and Clear-Lens□ Pro (allavailable from Novo Nordisk A/S, Bagsvaerd, Den.).

[0080] Other commercially available proteases include Maxatase□,Maxacal□, Maxapem□, Opticlean□ and Purafect□ (available from GenencorInternational Inc. or Gist-Brocades).

[0081] Examples of commercially available lipases include Lipolase□,Lipolase□ Ultra, LipoPrime, Lipozyme□, Palatase□, Novozym□ 435 andLecitase□ (all available from Novo Nordisk A/S).

[0082] Other commercially available lipases include Lumafast□(Pseudomonas mendocina lipase from Genencor International Inc.);Lipomax□ (Ps. pseudoalcaligenes lipase from Gist-brocades/Genencor Int.Inc.; and Bacillus sp. lipase from Solvay enzymes. Further lipases areavailable from other suppliers.

[0083] Examples of commercially available carbohydrases includeAlpha-Gal□, Bio-Feed□ Alpha, Bio-Feed□ Beta, Bio-Feed□ Plus, Bio-Feed□Plus, Novozyme□ 188, Celluclast□, Cellusoft□, Ceremyl□, Citrozym□,Denimax□, Dezyme□, Dextrozyme□, Finizym□, Fungamyl□, Gamanase□,Glucanex□, Lactozym□, Maltogenase□, Pentopan□, Pectinex□, Promozyme□,Pulpzyme□, Novamyl□, Termamyl□, AMG□ (Amyloglucosidase Novo),Maltogenase□, Sweetzyme□ and Aquazym□ (all available from Novo NordiskA/S). Further carbohydrases are available from other suppliers.

[0084] The amount of enzyme to be incorporated in a granule of theinvention will depend on the intended use of the granulate. For manyapplications, the enzyme content will be as high as possible orpracticable.

[0085] The content of enzyme (calculated as pure enzyme protein) in agranule of the invention will typically be in the range of from about0.5% to 20% by weight of the core.

[0086] When, for example, a protease (or peptidase) is incorporated ingranules according to the invention, the enzyme activity (proteolyticactivity) of the finished granules will typically be in the range of1-50 KiloNovoProteaseUnits per gram. Likewise, in the case of, forexample, Δ-amylases, an activity of 10-500 KiloNovoUnits per gram willbe typical, whilst for lipases, an activity in the range of 50-400KiloLipolaseUnits per gram will normally be suitable. All units areknown to the art.

[0087] Additional Coatings

[0088] The granules of the present invention may comprise one, two ormore additional coating layers on the inside or outside surface of theprotective coating according to the invention.

[0089] The additional coating layers may perform any of a number offunctions in the granule, depending on the intended use of the granule.Thus, for example, an additional coating may achieve one or more of thefollowing effects:

[0090] (i) further reduction of the dust-formation tendency of a granulewithout the additional coating according to the invention;

[0091] (ii) further protection of enzyme(s) in the granule againstoxidation by bleaching substances/systems (e.g. perborates,percarbonates, organic peracids and the like);

[0092] (iii) dissolution at a desired rate upon introduction of thegranule into a liquid medium (such as an aqueous medium);

[0093] (iv) provide a better physical strength of the granule.

[0094] Any additional conventional coating(s) of desired properties maybe applied and examples of conventional coating materials and coatingmethods is, inter alia, described in U.S. Pat. No. 4,106,991, EP 170360,EP 304332, EP 304331, EP 458849, EP 458845, WO 97/39116, WO 92/12645, WO89/08695, WO 89/08694, WO 87/07292, WO 91/06638, WO 92/13030, WO93/07260, WO 93/07263, WO 96/38527, WO 96/16151, WO 97/23606, U.S. Pat.Nos. 5,324,649, 4,689,297, EP 206417, EP 193829, DE 4344215, DE 4322229A, DD 263790, JP 61162185 A, JP 58179492.

[0095] In appropriate embodiments of granules according to the presentinvention, the additional coating layer may be composed as described inU.S. Pat. No. 4,106,991 [e.g. with a waxy material such as polyethyleneglycol (PEG), optionally followed by powdering with a whitener such astitanium dioxide].

[0096] Additional coating layers may further comprise one or more of thefollowing: anti-oxidants, chlorine scavengers, plasticizers, pigments,lubricants (such as surfactants or antistatic agents) additional enzymesand fragrances.

[0097] Plasticizers useful in coating layers in the context of thepresent invention include, for example: polyols such as sugars, sugaralcohols, or polyethylene glycols (PEGs) having a molecular weight lessthan 1000; urea, phthalate esters such as dibutyl or dimethyl phthalate;and water.

[0098] Suitable pigments include, but are not limited to, finely dividedwhiteners, such as titanium dioxide or kaolin, coloured pigments, watersoluble colorants, as well as combinations of one or more pigments andwater soluble colorants.

[0099] As used in the present context, the term “lubricant” refers toany agent which reduces surface friction, lubricates the surface of thegranule, decreases tendency to build-up of static electricity, and/orreduces friability of the granules. Lubricants can also play a relatedrole in improving the coating process, by reducing the tackiness ofbinders in the coating. Thus, lubricants can serve as anti-agglomerationagents and wetting agents.

[0100] Examples of suitable lubricants are polyethylene glycols (PEGs)and ethoxylated fatty alcohols.

[0101] In an especially preferred embodiment of the invention, only alubricant is applied as additional coating. The composition of 1) anenzyme containing core, 2) a coating and 3) and an additional lubricantcoating has shown particularly good properties with respect to enzymestability.

[0102] Coating Methods

[0103] The invention also relates to a method for manufacture/productionof the enzyme containing granule described herein. Conventional coatingmethods may be used to apply the coating according to the invention asdescribed by the references stated in the previous section (above).

[0104] A method for production of the enzyme containing granule maycomprise the following steps:

[0105] a) mixing an enzyme containing core material with a liquid mediumcomprising the water soluble compound of the invention and,

[0106] b) removing volatile components of the liquid medium from themixture, so as to deposit the nonvolatile components of the liquidmedium as solid coating layer on the core material.

[0107] In a preferred embodiment of the invention the enzyme containinggranule is produced by a fluid bed process comprising:

[0108] a) fluidising an enzyme containing core material in a fluid bedapparatus,

[0109] b) introducing a liquid medium comprising the water solublecompound of the invention by atomization of the liquid medium into thefluid bed, so as to deposit nonvolatile components of the liquid mediumas a solid coating layer on the core material and,

[0110] c) removing volatile components of the liquid medium from thecoated core material.

[0111] In a further preferred embodiment of the invention the corematerial is prepared by a method comprising:

[0112] a) Preparing a particulate carrier material,

[0113] b) introducing a liquid medium comprising an enzyme byatomization of the liquid medium into the fluid bed, so as to depositnonvolatile components including the enzyme of the liquid medium as anenzyme containing layer on the carrier, and

[0114] c) removing volatile components of the liquid medium from thecore material.

[0115] The particulate carrier material may in a preferred embodimentcomprise a binder (such as Glucidex□ 21D, from Roquette Freres), a fibrematerial (such as cellulose fibres) and a filler (such as finely groundsodium sulfate and/or kaolin). The particulate carrier may as well beprepared/granulated and dried as described in Example 1 in U.S. Pat. No.4,106,991. Following granulation the dry particulate carrier maysuitably be sieved, and fractionated after size to obtain a uniformcarrier size. Preferred carrier sizes measured as the diameter of thecarrier are between 0.1-2 mm, e.g. 0.3-1.0 m

[0116] As a further preferred embodiment additional enzyme may beabsorbed on the particulate carrier prior to applying the enzyme layer(II) This absorption may be achieved by:

[0117] a) absorbing the enzyme(s) into the surface of the carrier bycontacting the particulate carrier with a liquid comprising the enzymein a mixer,

[0118] b) mixing the composition by means of mixing blades, and

[0119] c) drying the enzyme loaded carrier by fluidising it in a fluidbed apparatus,

[0120] Conventional mixing equipment can satisfactorily be used to mixthe particulate carrier with the enzyme-containing liquid medium. Themixing equipment can be a batch mixer or a continuous mixer, such as aconvective mixer see, e.g. , Harnby et al., Mixing in the ProcessIndustries, pp. 39-53 (ISBN 0-408-11574-2)]. Non-convective mixingequipment, e.g. rotating drum mixers or so-called pan-granulators, mayalso be employed.

[0121] Drying of enzyme-loaded particulate carrier, application of theenzyme containing layer (II) the coating (III) and any additionalcoatings may be performed in any type of fluidising equipment (such asin a fluid-bed apparatus or other form of fluidizing equipment, such asa Hütttlin-type fluidizer). For a description of suitable fluid-bedequipment, see, e.g., Harnby et al., Mixing in the Process Industries,pp. 54-77 (ISBN 0-408-11574-2).

[0122] Applications of the Enzyme Containing Granule

[0123] The enzyme containing granule according to the invention isuseful where ever enzymes are to be stored alone or to be incorporatedin another dry product, and an improved enzyme stability is needed toenable good storage properties (improved shelf life) of the granule.Especially at relatively humid conditions, i.e. under an atmosphere witha % RH of more than 55% RH, preferably more than 60% RH, e.g. more than70% RH. Especially at conditions with more than 75% RH, more than 85% RHor more than 95% RH the invention is useful. The granule is alsoparticularly useful in dry products comprising oxidative compounds suchas peroxides or superoxides, e.g. bleach (e.g. perborates orpercarbonates) or other reactive components, which in case of contactwith the enzyme is able of inactivating the enzyme. Thus the inventionprovides a detergent composition comprising the granule of theinvention. The enzyme containing granule is further useful for cleaningan object (e.g. textile of cotton or other natural or synthetic fabrics)by contacting the object with an aqueous solution of the enzymecontaining granule. Finally the enzyme containing granule is useful inproducts such as animal feed/fodder or bakers flour.

[0124] Detergent Disclosure

[0125] A detergent composition of the invention comprises the enzymecontaining granule of the invention and a surfactant. Additionally, itmay optionally comprise a builder, another enzyme, a suds suppresser, asoftening agent, a dye-transfer inhibiting agent and other componentsconventionally used in detergents such as soil-suspending agents,soil-releasing agents, optical brighteners, abrasives, bactericides,tarnish inhibitors, coloring agents, and/or encapsulated ornon-encapsulated perfumes.

[0126] The detergent composition according to the invention can be inbars or granular forms. The pH (measured in aqueous solution at useconcentration) will usually be neutral or alkaline, e.g. in the range of7-11.

[0127] An enzyme contained in the granule of the invention incorporatedin the detergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01% to 0.2% of enzyme protein by weight ofthe composition.

[0128] Surfactant System

[0129] The surfactant system may comprise nonionic, anionic, cationic,ampholytic, and/or zwitterionic surfactants. The surfactant systempreferably consists of anionic surfactant or a combination of anionicand nonionic surfactant, e.g. 50-100% of anionic surfactant and 0-50%nonionic. The laundry detergent compositions may also contain cationic,ampholytic, zwitterionic, and semi-polar surfactants, as well as thenonionic and/or anionic surfactants other than those already describedherein.

[0130] The surfactant is typically present at a level from 0.1% to 60%by weight. Some examples of surfactants are described below.

[0131] a) Nonionic Surfactant

[0132] The surfactant may comprise polyalkylene oxide (e.g. polyethyleneoxide) condensates of alkyl phenols. The alkyl group may contain fromabout 6 to about 14 carbon atoms, in a straight chain or branched-chain.The ethylene oxide may be present in an amount equal to from about 2 toabout 25 moles per mole of alkyl phenol.

[0133] The surfactant may also comprise condensation products of primaryand secondary aliphatic alcohols with about 1 to about 25 moles ofethylene oxide. The alkyl chain of the aliphatic alcohol can either bestraight or branched, and generally contains from about 8 to about 22carbon atoms.

[0134] Further, the nonionic surfactant may comprise polyethylene oxidecondensates of alkyl phenols, condensation products of primary andsecondary aliphatic alcohols with from about 1 to about 25 moles ofethylene oxide, alkylpolysaccharides, and mixtures hereof. Mostpreferred are C8-C14 alkyl phenol ethoxylates having from 3 to 15 ethoxygroups and C8-C18 alcohol ethoxylates (preferably C10 avg.) having from2 to 10 ethoxy groups, and mixtures thereof.

[0135] b) Anionic Surfactants

[0136] Suitable anionic surfactants include the alkyl sulfatesurfactants which are water soluble salts or acids of the formula ROSO3Mwherein R preferably is a C10-C24 hydrocarbyl, preferably an alkyl orhydroxyalkyl having a C10-C20 alkyl component, more preferably a C12-C18alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metalcation (e.g. sodium, potassium, lithium), or ammonium or substitutedammonium. Other anionic surfactants include salts (including, forexample, sodium, potassium, ammonium, and substituted ammonium saltssuch as mono- di- and triethanolamine salts) of soap, C8-C22 primary orsecondary alkanesulfonates, C8-C24 olefinsulfonates, sulfonatedpolycarboxylic acids prepared by sulfonation of the pyrolyzed product ofalkaline earth metal citrates.

[0137] Alkylbenzene sulfonates are suitable, especially linear(straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl grouppreferably contains from 10 to 18 carbon atoms. The laundry detergentcompositions typically comprise from about 1% to about 40%, preferablyfrom about 3% to about 20% by weight of such anionic surfactants.

[0138] Builder System

[0139] The compositions according to the present invention may furthercomprise a builder system. Any conventional builder system is suitablefor use herein including aluminosilicate materials, silicates,polycarboxylates and fatty acids, materials such as ethylenediaminetetraacetate (EDTA), metal ion sequestrants such asaminopolyphosphonates. Phosphate builders can also be used herein.

[0140] Suitable builders can be an inorganic ion exchange material,commonly an inorganic hydrated aluminosilicate material, moreparticularly a hydrated synthetic zeolite such as hydrated zeolite A, X,B, HS or MAP. Detergency builder salts are normally included in amountsof from 5% to 80% by weight of the composition. Preferred levels ofbuilder for liquid detergents are from 5% to 30%.

[0141] Bleaching Agents

[0142] The detergent composition may also comprise a bleaching agents,e.g. an oxygen bleach or a halogen bleach. The oxygen bleach may be ahydrogen peroxide releasing agent such as a perborate (e.g. PB1 or PB4)or a percarbonate, or it may e.g. be a percarboxylic acid. The particlesize of a bleaching agent may be 400-800 microns. When present, oxygenbleaching compounds will typically be present at levels of from about 1%to about 25%.

[0143] The hydrogen peroxide releasing agent can be used in combinationwith bleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzene-sulfonate (NOBS),3,5-trimethyl-hexsanoloxybenzene-sulfonate (ISONOBS) orpentaacetylglucose (PAG).

[0144] The halogen bleach may be, e.g. a hypohalite bleaching agent, forexample, trichloro-isocyanuric acid and the sodium and potassium salt ofdichloroisocyanurates and N-chloro and N-bromo alkane sulfonamides. Suchmaterials are normally added at 0.5-10% by weight of the finishedproduct, preferably 1-5% by weight.

[0145] Granular detergent compositions according to the presentinvention can also be in “compact form”, i. e. they may have arelatively higher density than conventional granular detergents, i.e.form 550 to 950 g/l.

[0146] The compositions of the invention may for example, be formulatedas hand and machine laundry detergent compositions including laundryadditive compositions and compositions suitable for use in thepretreatment of stained fabrics, rinse added fabric softenercompositions, and compositions for use in general household hard surfacecleaning operations and dishwashing operations.

[0147] More specifically, the enzyme containing granules of theinvention may be incorporated in the detergent compositions described inWO 97/04079, WO 97/07202, WO 97/41212, and PCT/DK 97/00345.

Materials and Methods

[0148] Preparation of Carrier Material

[0149] Portions of carrier material was prepared by spraying 3.0 kg offibrous cellulose (Arbocel□ FTC200), 3.0 kg of kaolin (Speswhite□,English China Clay) and 20.5 kg of finely ground sodium sulfate with 9.5kg of a 21% w/w aqueous solution of carbohydrate binder (Glucidex□ 21D,from Roquette Freres). This mixtures was granulated and dried asdescribed in Example 1 in U.S. Pat. No. 4,106,991. The dry granulatedcarrier material was sieved, and the fraction between 0.3 and 1.0 mm wasseparated and used for further processing.

[0150] Absorbing Enzyme on the Carrier Material

[0151] The granulated and fractionated carrier material was transferredto a Lodiger mixer equipped with a multiple chopper head, and sprayedwith a liquid Savinase™ enzyme concentrate solution approximately 33KNPU/g containing 0.4% w/w g of a nonionic surfactant (Softanol 50). Theenzyme containing solution was sprayet onto the carrier at dosage of0.15 kg solution per kg carrier material. The nonionic surfactant wasadded to the enzyme concentrate to enhance the absorption of the enzymeonto the carrier. The Savinase□ concentrate was applied using a pressurenozzle submerged in the carrier and spraying directly into the chopper.The carrier and the Savinase™ concentrate was mixed by means of mixingblades and the mixing blades and the chopper were operated continuouslyduring the spraying. The resulting product was transferred to a GlattWSG 15 fluid-bed apparatus (Glatt, Germany) with an air-inlettemperature of 62θC., and dried for 30 minutes, or until the producttemperature exceeded 50θC., and then sieved on a 1.2 mm mesh screen,leaving only 0.8% w/w of residual, oversized particles on the sieve.

[0152] Measurement of Enzyme Stability

[0153] For each type of enzyme containing granule samples of thegranules are mixed with a commercial detergent. One or of these samplesare immediately sealed in glass jars and stored below −18° C. Thesesamples are reference samples and the enzyme herein is by definition100% stabile. Other samples are placed in climate controlled cupboardsand stored for a preset period of time in open glass jars at differenttemperatures and humidities (% RH). When a preset storage period isfinished samples are removed from the “climate static” conditions, andthe glass jars immediately sealed and cooled to below −18° C. to stopany progressing inactivation of the enzyme. When all samples has beenstored for the preset period of time, all samples including thereference samples are analyzed the day in an appropriate enzyme activityassay and the test results of the samples are calculated as percent ofthe reference sample test results.

[0154] Enzyme Assay

[0155] The enzyme assay used herein is a protease activity assay, andthe unit for protease activity herein is Kilo Novo Protease Units pergram of sample (KNPU/g). The activity is determined relatively to anenzyme standard (Savinase™) of known activity. The enzyme standard isstandardized by measuring for a given amount of enzyme the formationrate (μmol/minute) of free amino groups liberated from digestion ofdi-methyl-casein (DMC) in solution by the enzyme. The formation rate ismonitored by recording the linear development of absorbance at 420 nm ofthe simultaneous reaction between the formed free amino groups and added2,4,6-tri-nitro-benzene-sulfonic acid (TNBS). The digestion of DMC andthe color reaction is carried out at 50° C. in a pH 8.3 boric acidbuffer with a 9 mn. reaction time followed by a 3 min. measuring time. Afolder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark,which folder is hereby included by reference.

[0156] For samples of the enzyme containing detergents and thestandardized enzyme standard a modified assay was used, wherein thereaction was carried out at 40° C. in a pH 8.3 boric acid buffercontaining 3.1 g/L boric acid (Merck), 11.18 g/L potassium chloride(Merck), 1.5 mL/L 15% BriJ 35 (Merck) and 20 g/L sodium sulfite bleachscavenger.

[0157] For measuring Endolase activity (a cellulase) any conventionalendo cellulose viscosity reduction method may be used (such as themethod described in B1087a-GB available upon request from Novo NordiskA/S-Denmark). Endolase digests CMC (carboxy-methyl-cellulose) insolution thereby reducing the viscosity of the solution and theviscosity reduction is related to the activity of the endolase.

[0158] For measuring alfa-amylase activity (e.g. Natalase®) anyconventional alfa-amylase assay may be used (such as the methoddescribed in AF318/1-GB available upon request from Novo NordiskA/S-Denmark). Alfa-amylases cleaves 1-6 alfa bonds between adjacentglucose units. By using e.g. a 2-chlor-4-nitrophenyl-b-D-maltoheptaosidsubstrate and alfa- and beta-glucosidase enzymes the substrae may becompletely digested into monosaccharides and 2-chlor-4-nitrophenol,which form a detectable colour. Kits for performing these assays arecommercially available.

[0159] Measurement of Constant Humidity Values for Coating Compounds

[0160] The constant humidity of water soluble compound coating materialswere measured by preparing a saturated aqueous solution with excess ofsolid phase of the compound in an open beaker. After equilibrium hasbeen reached and no more of the compound dissolves in the liquid phase,and solid particles of the compound are still visible, the beaker isplaced in a sealed thermostated humidity measuring device (e.g. aNovasina apparatus), which measures the % RH at the chosen temperatureof the atmosphere over the saturated solution.

EXAMPLES

[0161] The invention disclosed herein is illustrated by the non-limitingexamples given below

Example 1

[0162] A sample of the dry enzyme-loaded granular carrier wastransferred to a Glatt WSG5 conventional fluid bed apparatus. Using aconventional top spray coating technique with an air inlet temperatureof 70° C., air outlet temperature of 42° C. and with air volume of 600m3/h the following steps were carried out in sequence:

[0163] a) an enzyme containing layer was applied onto the carrier byspraying an enzyme containing aqueous solution containing Savinase™,PVP/VA co-polymer (Luviscol VA64) and titaniumdioxide (TiO₂) onto thecarrier at a spraying rate for the enzyme solution of 100 g/min.Approximately 210 g Savinase™ concentrate (30 KNPU/g), 2.34 g of PVP/VAco-polymer (Luviscol VA64) and 2.5 g of titaniumdioxide (TiO₂) wereapplied per kg carrier,

[0164] b) a 80 g sodium citrate per kg carrier coating layer of wasapplied by spraying a 37% w/w aqueous solution of sodium citrate ontothe product of a), at a spraying rate for the coating solution of 100g/min,

[0165] c) an additional coating layer of 50 g titaniumdioxide (Kronos2044), 50 g kaolin (ECC Supreme), 55 g Glascol LS27 (46% suspension fromAllied Colloids Ltd.-GB), 30 g PEG 4000 and 1.67 g Softanol 50 per kgcarrier was applied by spraying an aqueous solution of these componentsonto the product of b), at a spraying rate for the coating solution of100 g/min, and

[0166] d) a final coating was applied by spraying a solution of 7.5 gPEG 4000 per kg carrier onto the product of c), at a spraying rate forthe coating solution of 100 g/min.

[0167] The finished enzyme containing granule was dried for 5 minutesand then cooled to 30° C., where after it was removed from the fluid bedand sifted between 300 and 1200 μm

[0168] As a reference two different types of granules were prepared. Thefirst type of granule was prepared by repeating steps a)-d) with theexception that the coating described in b) was replaced by a 80 gammonium sulfate per kg carrier coating layer by spraying a 37% w/waqueous solution of ammonium sulfate onto the product of a), at aspraying rate for the coating solution of 100 g/min. The second type ofgranule was also prepared by repeating steps a)-d), but with theexception that the coating described in b) was replaced by a 80 g sodiumformiate per kg carrier coating layer by spraying a 37% w/w aqueoussolution of sodium formiate onto the product of a), at a spraying ratefor the coating solution of 100 g/min.

Example 2

[0169] The storage stability of the enzyme containing granulates fromExample 1 were tested in one powder detergent (A) containing perboratebleach and TAED and one powder detergent (B) containing percarbonatebleach. 100 mg samples of the enzyme containing granulates and 10.0 gsamples of the detergents were mixed to constitute test samples. Thesamples were incubated in open jars at air conditions 35° C. and 55% RHin a thermostated and humidity controlled cupboard. Identical sampleswere taken out from the cupboard after 2 and 4 weeks and analyzed forenzyme (Savinase™) activity together with identical reference samples,which had been stored in sealed jars below −18°0 C. The results aregiven in Example 1, wherein the enzyme activities of the samples aregiven as a percentage of the corresponding reference samples. TABLE 1Residual activity Residual activity Coating in Detergent A in DetergentB compound 2 weeks 4 weeks 2 weeks 4 weeks Invention Sodium 76% 51% 82%69% granule citrate Reference Ammonium 56% 39% 70% 59% granule sulfateReference Sodium 35% 21% 32% 21% granule formiate

[0170] Constant humidities at 25° C.: Ammonium sulfate=79; sodiumformiate=56 and sodium citrate=86. The results show that the coatingwith highest constant humidity provides the best storage stability ofthe enzyme.

Example 3

[0171] The hygroscopicity of the samples from Example 1 were testedincluding a reference sample which was identical to the granulates ofExamples 1 to 3 except that it did not contain any salt coating ofammonium sulfate, sodium citrate or sodium formiate. All samples wereincubated for 1 week in open jars at 3 different conditions: 55% RH and35° C., 60% RH and 30° C. and 74% RH and 37° C. Subsequently the waterabsorption of the samples were determined by weighing the samples beforeand after the incubation. Further after the incubation the samples wereexamined under a microscope. The results are given in Table 2, whichshows the water absorption of the samples at different humidities. Theseresults clearly shows big difference in how much water is absorbed inidentical granules with different coatings, indicating that sodiumcitrate has the lowest water absorbing properties (the highest constanthumidity value). For comparison a sample of a granulate with a differentcore material and a coating of ammonium sulfate is included (PurafectG). This result shows that if the coating allows moisture to pass, thecore may absorb huge amounts of water. Table 2 further lists the resultsof the microscope examination, which showed that the coating of some ofthe samples were damaged (cracks had developed) which was most clearlyobserved when the water absorption of the coating (absorption of thesample minus the absorption of the reference) had reached a level ofapproximately 50% of the salt layer weight (which constitutes 6% oftotal formula). TABLE 2 % water absorption Coating 55% 60% 70% Coatingdamage Sample (III) RH RH RH observed at Core no salt 1.1 1.7 2.8 Nodamage material coating reference Ammonium 1.4 2.2 6.1 70% RH granulesulfate invention Sodium 1.2 1.9 3.4 No damage granule citrate ReferenceSodium 4.8 7.3 12.3 60% RH granule formiate Purafect G Commercial 0.91.9 23.2 Liquefies at product 70% RH

[0172] Constant humidities at 25° C.: Ammonium sulfate=79; sodiumformiate=56 and sodium citrate=86.

Example 4

[0173] An enzyme containing granule coated with sodium citrate wasproduced as in Example 1 with the exception that the pH of the sodiumcitrate was adjusted to 7.5 by adding citric acid to the coatingsolution prior to the coating process.

Example 5

[0174] An enzyme containing granule was produced as in Example 4 withthe exception that the coating of pure sodium citrate was replaced by amixture of sodium citrate and sodium thiosulfate in the weight ratio9:1. Thus a coating consisting of 72 g sodium citrate and 8 gthiosulfate per kg carrier was applied.

Example 6

[0175] An enzyme containing granule was produced as in Example 4 withthe exception that the coating of pure sodium citrate was replaced by amixture of sodium citrate and sodium thiosulfate in the weight ratio4:1. Thus a coating consisting of 64 g sodium citrate and 16 gthiosulfate per kg carrier was applied.

Example 7

[0176] The storage stability of the enzyme containing granules fromExamples 4-6 as well as the ammonium sulfate reference granule ofExample 1 were tested in one powder detergent (A) containing perboratebleach and TAED and one powder detergent (B) containing percarbonatebleach. 100 mg samples of the enzyme containing granulates (Purafect 50mg) and 10.0 g samples of the detergents were mixed to constitute testsamples. The samples were incubated in open jars at air conditions 35°C. and 55% RH in a thermostated and humidity controlled cupboard.Identical samples were taken out from the cupboard after 2 and 4 weeksand analyzed for enzyme (Savinase™) activity together with identicalreference samples, which had been stored in sealed jars below −18° C.The results are given in Example 3, wherein the enzyme activities of thesamples are given as a percentage of the corresponding referencesamples. TABLE 3 Residual activity Residual activity Coating inDetergent A in Detergent B compound 2 weeks 4 weeks 2 weeks 4 weeksReference ammonium 53% 36% 73% 61% granule of sulfate (Ex. 1) InventionSodium 80% 51% 74% 68% granule citrate pH 7.5 Invention 90% Sodium 96%78% 90% 92% granule citrate, 10% thiosulfate Invention 80% Sodium 103% 89% 95% 92% granule citrate, 20% thiosulfate Purafect G Commercial 89%79% 90% 81% product

[0177] Constant humidities at 25° C.: Ammonium sulfate=79 and sodiumcitrate=86.

Example 8

[0178] An enzyme containing granule was produced as in Example 1 withthe exception that i) the enzyme solution used for both absorption ofenzyme onto the carrier and for applying the enzyme layer containedsodium thiosulfate in an amount corresponding to 0.33% w/w of the finalcoated granulate and ii) the sodium citrate coating was replaced by a 80g per kg carrier sodium sulfate coating by applying a 45° C., 28.6% w/waqueous sodium sulfate solution.

[0179] Further a reference granule was prepared identical to the firstgranule in this example with the exception that the sodium sulfatecoating was replaced with a 80 g per kg carrier coating of ammoniumsulfate.

Example 9

[0180] The storage stability of the enzyme containing granules fromExample 8 were tested along with a commercial product in one powderdetergent (A) containing sodium perborate bleach and TAED, one powderdetergent (B) containing sodium percarbonate bleach and one powderdetergent (C) without bleach. 100 mg samples (50 mg Purafect G) of theenzyme containing granulates and 10.0 g samples of the detergents weremixed to constitute test samples. The bleach containing samples wereincubated in open jars at air conditions 35° C. and 55% RH, while thesamples without bleach were incubated in open jars at air conditions 37°C. and 70% RH. All samples were incubated in thermostated and humiditycontrolled cupboards. Identical samples were taken out from the cupboardafter 2 and 4 weeks and analyzed for enzyme (Savinase™) activitytogether with identical reference samples, which had been stored insealed jars below −18° C. The results are given in Table 4, wherein theenzyme activities of the samples are given as a percentage of thecorresponding reference samples. TABLE 4 Coating compound 2 weeks 4weeks 2 weeks 4 weeks Residual activity Residual activity in Detergent Ain Detergent B Reference ammonium 68% 49% 87% 78% granule sulfateInvention Sodium 90% 72% 98% 100%  granule sulfate Purafect G commercial92% 83% product Residual activity in Detergent C Reference ammonium 74%53% granule sulfate Invention Sodium 93% 92% granule sulfate Purafect GCommercial 83% 47% product

[0181] Constant humidities at 25° C.: Ammonium sulfate=79; sodiumsulfate−93.

[0182] The results show that coating materials with higher constanthumidity results in improved stability.

Example 10

[0183] An enzyme containing granule coated with sodium sulfate wasproduced as in Example 8 with the exception that a different ultrafiltrated Savinase™ concentrate solution (app 41 KNPU/g) was usedthroughout the process.

Example 11

[0184] A sample of dry enzyme-loaded granular carrier was produced asdescribed vide supra with the exception that the enzyme solution usedfor absorbing enzyme onto the carrier was added 5.33 g of sodiumthiosulfate per kg finished dry carrier. The enzyme and thiosulfateloaded carrier was transferred to a Glatt WSG5 conventional fluid bedapparatus. Using a conventional top spray coating technique with an airinlet temperature of 70° C., air outlet temperature of 42° C. and withair volume of 600 m3/h the following steps were carried out in sequence:

[0185] a) an enzyme containing layer was applied onto the carrier byspraying an enzyme containing aqueous solution containing Savinase™,PVP/VA co-polymer (Luviscol VA64), titaniumdioxide (TiO₂) and sodiumthiosulfate onto the carrier at a spraying rate of 100 g/min.Approximately 87 g Savinase™ concentrate (41 KNPU/g), 2.67 g of PVP/VAco-polymer (Luviscol VA64), 1.67 g of titaniumdioxide (TiO₂) and 1.67 gsodium thiosulfate were applied per kg carrier,

[0186] b) a 267 g sodium sulfate per kg carrier coating layer of sodiumsulfate was applied by spraying a 28.5% w/w aqueous solution of sodiumsulfate at approximately 45° C. onto the product of a), at a sprayingrate for the coating solution of 100 g/min,

[0187] c) a final lubricant coating was applied by spraying a solutionof 7.33 g PEG 4000 per kg carrier onto the product of c), at a sprayingrate of 100 g/min.

[0188] The finished enzyme containing granulate was dried for 5 minutesand then cooled to 30° C., where after it was removed from the fluid bedand sifted between 300 and 1200 μm

Example 12

[0189] The storage stability of the enzyme containing granulates fromExamples 13 and 14 were tested along with two commercial products in onepowder detergent (A) containing perborate bleach and TAED, one powderdetergent (B) containing percarbonate bleach and one powder detergent(C) without bleach. 150 mg samples of the enzyme containing granulates(75 mg Purafect E and G) and 10.0 g samples of the detergents were mixedto constitute test samples. The bleach containing samples were incubatedin open jars at air conditions 35° C. and 55% RH, while the sampleswithout bleach were incubated in open jars at air conditions 37° C. and70% RH. All samples were incubated in thermostated and humiditycontrolled cupboards. Identical samples were taken out from the cupboardafter 2 and 4 weeks and analyzed for enzyme (Savinase™) activitytogether with identical reference samples, which had been stored insealed jars below −18° C. The results are given in Table 5, wherein theenzyme activities of the samples are given as a percentage of thecorresponding reference samples. TABLE 5 Coating compound 2 weeks 4weeks 2 weeks 4 weeks Residual activity Residual activity in Detergent Ain Detergent B Invention Sodium 82% 55% 85% 77% granule sulfate (Ex. 10)Invention Sodium 98% 98% 96% 96% granule sulfate (Ex. 11) Purafect Bcommercial 88% 65% 51% 41% product Purafect G commercial 95% 75% 73% 62%product Residual activity in Detergent C Invention sodium 94% 80%granule sulfate (Ex. 10) Invention sodium 99% 95% granule sulfate (ex.11) Purafect E commercial 65% 17% product Purafect G Commercial — —product

[0190] The results given in Table 5 shows that application of a thickcoating of with a high constant humidity leads to superior stabilityeven if only a lubricant is applied as an additional coating.

Example 13

[0191] 20 kg of uncoated Savinase® (protease enzyme) containinggranulate (type TX) was produced as described in U.S. Pat. No. 4,106,991Example 1 with the following exceptions:

[0192] 1) sodium sulfate was used in stead of sodium chloride as fillermaterial

[0193] 2) the enzyme concentrate was an aqueous suspension ofcrystalline enzyme containing also a binder (Glucidex) and 0.4% w/wmethionine as an antioxidant

[0194] The uncoated enzyme granule was transferred to a 50 litre Lödigemixer and sprayed with 2.0 kg of a solution/suspension consisting of 25%sodium sulfate, 12% dextrine, 7% TiO2 and 56% of water. The granularmass was during the spraying treated with the chopper as described inU.S. Pat. No. 4,106,991.

[0195] The mixer treated granulate was subsequently transferred to aGlatt WSG5 fluid bed and dried. 15 kg of the dried granulate wasthereafter successively coated in three steps with parameters asdescribed in Example 1.

[0196] a) the enzyme containing granulate was in a first step coatedwith 11.0 kg of a solution/suspension consisting of 27.1% sodium sulfate3.9% TiO2, 1.0% dextrin and water ad 100%. The salt solution was duringthe spraying kept at 45-50° C. to avoid crystallisation of the sodiumsulfate. The coated the enzyme containing granule with a coatingcontaining 85% w/w sodium sulfate.

[0197] b) an additional coating was applied as a dust suppressing filmby spraying with 3.5 kg of a solution consisting of 6.3%methylhydroxypropyl cellulose (Aqualon 8MP5C) 6.3% PEG 4000 and water ad100%

[0198] c) the granulate was in a final step sprayed with 0.46 kg of a24% PEG 4000/water solution.

Example 14

[0199] A comparative base granulate was prepared as described in Example13 without the mixer treatment and without fluid bed coatings and wasconventionally coated as described in U.S. Pat No. 106,991 Example 22 byapplying a solution of 7% PEG 4000 and 12.5% of a 1:1 TiO2/Kaolinmixture.

Example 15

[0200] The storage stability of granulates of Examples 13 and 14 wastested in a perborate containing (A) and a percarbonate containing (B)detergent as described in Example 4. TABLE 6 Residual activity Residualactivity Coating in Detergent A in Detergent B compound 2 weeks 4 week 2weeks 4 weeks Example 13 Sodium 98 92 103  99 granulate sulfate Example14 comparative 69 43 63 48 granulate granule Purafect G Commercial 90 7397 83 product

Example 16

[0201] 15 kg of dry Savinase enzyme absorbed on a granular carrier wastransferred to a Glatt WSG conventional fluid bed apparatus. Using aconventional top spray coating technique with an air inlet temperatureof 70° C. and an air outlet of 42° C. and with an air volume air volumeof 600 m³/h were the following steps carried out in sequence:

[0202] a) an enzyme containing layer was applied onto the carrier byspraying granular carrier with an enzyme containing solution consistingof 2.07 kg of a liquid Savinase concentrate (82% dry matter, 24 KNPU/g),50 g Glucidex 21D and 54 g TiO2 and with a spraying rate of 100 g/min.

[0203] b) The salt layer was applied in a second step by spraying with14.7 kg of a solution consisting of 27% sodium sulfate, 3.9% TiO2, 1.0%Glucidex 21D and water ad 100%. The temperature of the solution was keptat 45-50° C. to avoid crystallisation of the salt.

[0204] c) The salt coated granulate was in a next step coated with adust suppressing film by spraying with 3.4 kg of a solution consistingof 6.3% methylhydroxypropyl cellulose, 6.3% PEG 4000 and water ad 100%.

[0205] d) the granulate was in a final step sprayed with 0.46 kg of a24% PEG 4000/water solution.

[0206] All enzyme concentrate used for this preparation had an additionof sodium thiosulfate to the concentrate corresponding to 0.3% w/w ofthe final granulate.

Example 17

[0207] This example was prepared as Example 16 with the exception thatthe solution for the coating (b) was reduced to 11.0 kg.

Example 18

[0208] This example was prepared as Example 16 with the exception thatthe solution for the coating (b) was reduced to 7.35 kg.

Example 19

[0209] This example was produced according to Example 16 step (a), i.e.without the salt coating and the dust suppressing films

Example 20

[0210] The storage stability of granulates of Examples 16-19 was testedand compared to references in three detergents as described in Example9. TABLE 7 Residual Residual activity (%) activity (%) Coating indetergent A in detergent B compound 2 weeks 4 weeks 2 weeks 4 weeksExample 16 Sodium sulfate 99 99 98 98 Example 17 Sodium sulfate 100  9694 97 (75% relative to Example 16) Example 18 Sodium sulfate 97 94 96 97(50% relative to Example 16) Example 19 Comparative 60 44 55 43 granulewithout sodium sulfate Example 14 Comparative 69 43 63 48 granulePurafect G Commercial 90 73 97 83 product

[0211] Residual activity (%) in detergent C Coating compound 2 weeks 4week Example 16 Sodium sulfate 94 82 Example 17 Sodium sulfate 92 71(75% relative to Example 16) Example 18 Sodium sulfate 93 76 (50%relative to Example 16) Example 19 Comparative granule 79 49 withoutsodium sulfate Example 14 Comparative granule 88 62 Purafect GCommercial product 89 47

Example 21

[0212] An Endolase (cellulase enzyme) containing granulate was producedas described in U.S. Pat. No. 4,106,991 Example 1 with the followingexceptions:

[0213] a) The filler was sodium sulfate

[0214] b) The liquid enzyme concentrate was used as the granulatingliquid.

[0215] c) The granulate had furthermore an addition of 10% w/w of acarbohydrate binder and 0.5% w/w of sodium thiosulfate.

[0216] The granulate was conventionally coated as described in U.S. Pat.No. 106,991 Example 22 by applying a solution of 7.2% w/w PEG 4000 and13.0% w/w of a 1:1 mixture of TiO2 and kaolin.

Example 22

[0217] An Endolase (cellulase enzyme) containing granulate was producedas described in Example 21. This base granulate was coated according tothe steps (b)-(d) in Example 16 with the exception that 10.6 kg of thesalt solution was applied in step (b).

Example 23

[0218] The storage stability of granulates of Examples 21 and 22 wastested in a sodium perborate containing detergent (A) with conditions asdescribed in Example 9. TABLE 8 Residual activity (%) in detergent ACoating compound 1 week 3 weeks 5 weeks Example 21 Comparative granule97  28 23 Example 22 Sodium sulfate 100 70

Example 24

[0219] A Natalase® (an amylase enzyme) containing granulate was producedas described in U.S. Pat. No. 4,106,991 Example 1 with the followingexceptions:

[0220] 1) sodium sulfate was used in stead of sodium chloride as fillermaterial

[0221] 2) the enzyme concentrate was an aqueous suspension ofcrystalline enzyme which was also used as the granulating liquidcontaining also a binder (Glucidex).

[0222] 3) The granulate had furthermore an addition of 0.4% w/w sodiumthiosulfate (calculated as % of the uncoated granulate).

[0223] The granulate was conventionally coated as described in U.S. Pat.No. 106,991 Example 22 by applying a solution of 7% of PEG 4000 and12.5% of a 1: 1 mixture of TiO2 and kaolin.

Example 25

[0224] A Natalase® containing granulate was produced as described inExample 24. 1300 g of the Natalase containing base granulate wastransferred to a UniGlatt fluid bed where coated with a salt coating byspraying it with a 50° C. salt solution at consisting of:

[0225] 234 g of sodium sulfate

[0226] 9 g of Glucidex 21D

[0227] 25 g of TiO2

[0228] 585 g of water

[0229] The spraying conditions were air inlet temperature 70° C. and airoutlet temperature 42° C. The granulate was after finishing the sprayingfurther dried in the fluid bed for 5 minutes

Example 26

[0230] The storage stability of granulates of Examples 24 and 25 wastested in the percarbonate containing detergent (B) with conditions asdescribed in Example 9. TABLE 9 Residual activity in detergent B Coatingcompound 1 week 2 weeks 4 weeks Example 24 Comparative granule 82 79 69Example 25 Sodium sulfate 98 97 94

Example 27

[0231] A non-coated Savinase® containing granulate (granulate A) wasproduced as described in U.S. Pat. No. 4,106,991 Example 1 wit thefollowing exceptions:

[0232] 1) sodium sulfate was used in stead of sodium chloride as fillermaterial

[0233] 2) the enzyme concentrate was an aqueous suspension ofcrystalline enzyme containing also a carbohydrate binder (Glucidex) andmethionine as an antioxidant

[0234] The non-coated granules was coated with a salt layer using afluid bed according to the following procedure:

[0235] a) 15 kg of the uncoated granules were fluidized in a Glatt WSG-5fluid bed using 550 m³ air per hour. The air inlet temperature was 70°C.

[0236] b) A salt solution of 2.0 kg Na₂SO₄ dissolved in 5.0 kg water at50° C. was prepared. 2.1 kg of this solution was sprayed onto thefluidized granules at a rate of 100 grams solution per minute. Duringspraying of the liquid the product temperature was approximately 42° C.After adding the solution the water was allowed to evaporate from thecoated granules (until the product temperature raised quickly in thefluid bed). A sample of 2.0 kg (granulate A1) of the coated granules wastaken out and the coating process was repeated to add further coating tothe granules remaining in the fluid bed by spraying the remaininggranules with an additional 1.75 kg of the salt solution. A sample of2.0 kg of the additionally coated granules (granulate A2) was taken outand the coating process was repeated once more to add even furthercoating to the granules remaining in the fluid bed by spraying theremaining granules with the remaining 3.15 kg of the salt solution(granulate A3).

Example 28

[0237] The enzyme stability in the granules were tested in a modelpowder detergent containing Sodium Perborate at 35° C. and 55% relativehumidity in open boxes according to Example 9. TABLE 10 ResidualSavinase activity in % Granulate 2 weeks 4 weeks Uncoated Savinase ®granules 47 36 A1 (4.0% Na₂SO₄) 68 42 A1 (7.8% Na₂SO₄) 77 55 A3 (15.8%Na₂SO₄) 92 75

[0238] The % salt was calculated as % w/w salt of the uncoated granulesunder the condition that all water was evaporated. From Example 10 itwas concluded that the enzyme stability in the Savinase® granules weresignificantly improved by the salt coating and that increasing theamount of salt improves the stability.

2. An enzyme containing granule comprising: a) an enzyme containing coreand b) a protective substantially continuous layer or coatingencapsulating the core comprising at least 60% of a water solublecompound, having a molecular weight below 500 grams per mole, a pH below11 and a constant humidity at 20° C. of more than 81%.
 3. The granuleaccording to claim 1, wherein said water soluble compound has amolecular weight between 30-500 g/mole.
 4. The granule according toclaim 2, wherein said water soluble compound has a molecular weightbetween 75-400 g/mole.
 5. The granule according to any preceding claim,wherein said is water soluble compound has a solubility of at least 0.1gram per 100 gram water.
 6. The granule according to any precedingclaim, wherein said water soluble compound has a solubility of at least10 gram per 100 gram water.
 7. The granule according to any precedingclaim, wherein said water soluble compound has constant humidity at 20°C. of more than 90%.
 8. The granule according to any preceding claim,wherein said core is a non absorbing core.
 9. The granule according toclaim 1, wherein said salt is selected from the group consisting ofalkali or earth alkali metal ion salt of sulfate, sulfite, phosphate,phosphonate, nitrate, chloride, carbonate and simple organic acids. 10.The granule according to claim 1, wherein said salt is selected from thegroup consisting of Na₂HPO₄, Na₃PO₄, (NH₄)H₂PO₄, KH₂PO₄, Na₂SO₄, K₂SO₄,KHSO₄, ZnSO₄ and sodium citrate.
 11. The granule according to anypreceding claim, wherein said coating further comprises one or moreprotective agents capable of inactivating components hostile to theenzyme entering the granule from a surrounding matrix.
 12. The granuleaccording to claim 10, wherein said hostile component is a detergentbleach component.
 13. The granule according to claim 11, wherein saidprotective agent is selected from the group consisting of reducingagents, antioxidants and salts of transition metals.
 14. The granuleaccording to claim 12, wherein said reducing agent is a salt ofthiosulfate.
 15. The granule according to any preceding claim, whereinsaid coating constitutes 1-75% w/w of the coated granule.
 16. Thegranule according any preceding claim, wherein said enzyme containingcore comprises a particulate carrier and an enzyme containing layer. 17.The granule according to claim 15, wherein additional enzyme is adsorbedinto the carrier.
 18. The granule according to claim 16, wherein saidabsorbed enzyme and/or enzyme containing layer comprises a protectivereducing agent.
 19. The granule according to any preceding claim,wherein said enzyme is selected from the group consisting ofoxidoreductases (EC 1.-.-.- ), transferases (EC 2-.-.-), hydrolases (EC3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-) and ligases (EC6.-.-.-).
 20. The granule according to any preceding claim furthercomprising an additional coating.
 21. The granule according to claim 19,wherein said additional coating is a lubricant.
 22. A method forproducing the granule of claims 1-20 comprising: a) mixing an enzymecontaining core material with a liquid medium comprising said watersoluble compound and, b) removing volatile components of the liquidmedium from the mixture, so as to deposit the nonvolatile components ofthe liquid medium as solid coating layer on the core material.
 23. Themethod according to claim 21 wherein the granule is obtained by a fluidbed process comprising: a) fluidising an enzyme containing core materialin a fluid bed apparatus, b) introducing a liquid medium comprising thewater soluble compound of the invention by atomization into the fluidbed, so as to deposit the nonvolatile components of the liquid medium assolid coating layer on the core material and, c) removing volatilecomponents of the liquid medium from the coated core material.
 24. Adetergent composition comprising the enzyme containing granularcomposition of any of the claims 1-20.
 25. A cleaning method comprisingcontacting an object with an aqueous solution comprising the particulatecomposition of any of the claims 1-20.